This application seeks continued support to complete the cloning and characterization of genes corresponding to the ARRESTED DEVELOPMENT (add) 1 and 3 mutations of Arabidopsis thaliana. The add1 mutation causes a temperature dependent loss of normal meristem function, cellular hypertrophy in the meristem, and differentiation of stem cells. These phenotypic characteristics of the mutant suggest that the wild-type allele of this gene may help establish and maintain the meristematic stem cell population at the shoot apex. The add3 mutation causes a temperature dependent loss of cell proliferation in the marginal regions of leaves, suggesting that this gene's normal role may be to stimulate cell proliferation in developmentally defined contexts in the leaf. In the past year wild-type DNA has been isolated from each ADD locus and been reintroduced into mutant backgrounds via Agrobacterium mediated plant genetic transformation. In both cases transformation rescue of the mutant phenotype has been observed, indicating that DNA including each gene has been identified. The cloning of these genes will facilitate identifying the genetic pathways and physiological context in which each gene acts. Because each gene has pronounced young seedling phenotypes, rt PCR and mRNA in situ hybridization analysis will be performed on mutant and wild-type seedlings to first determine the cell types in which these transcripts accumulate. Secondarily, the impact of each mutation will be assessed on the transcriptional regulation of known meristem regulators (the WUSCHEL, SHOO TMERISTEMLESS and CLAVATA 3 genes to begin with) and leaf developmental genes (ASYMMETRIC LEAVES 1, YABBY3 and FILAMENTOUS FLOWERS). The temperature sensitive aspect of both mutations will also be used to assay the response of the transcriptome via microarray as plants transition from more normal to severely affected phenotypes based on temperature change. These projects will support our long-term goal to understand the informational context required for meristem initiation and function, and to understand the regulatory interplay occurring between the meristem and developing leaves. [unreadable] [unreadable]